Recombinant human and animal adenoviruses are used extensively for their application in gene therapy and vaccination. For these applications, adenovirus vectors are used as carriers for a gene or genes of interest to be introduced into host cells. For example, adenovirus vectors can be used to express a gene or part thereof encoding a desired antigen to elicit an immune response.
First generation adenovirus vectors typically only included one transgene. Many strategies are published for these first generation vectors. The published strategies report the use of a variety of different adenovirus vectors and show that the transgene expression cassette can been placed in different regions of the adenovirus, for example in the E1 region, the E3 region, or between E4 and the right ITR.
For vaccine purposes, however, more than one antigen or the same antigen from several different strains is often required to achieve protection and broad coverage. Therefore, in certain cases, it is desirable to express at least two antigens from one adenoviral vector. Different approaches to encode two antigens in one adenoviral vector have been described.
In a first two antigen approach, one antigen expression cassette was placed in the E1 region and a second one was placed in the E3 region (e.g. (Vogels et al., 2007)). In a different two antigen approach, one antigen expression cassette was placed in E1 and a second one between E4 and the right ITR (e.g. (Holman et al., 2007; Pham et al., 2009; Schepp-Berglind et al., 2007)). In yet another two antigen approach, the two antigen expression cassettes were placed in the E1 region in a head-to-tail fashion using two different promoter sequences in an attempt to prevent genetic instability by recombination (e.g. (Belousova et al., 2006; Harro et al., 2009)).
Various other two antigen approaches have also been published for different viral vectors, for example with lentiviral vectors. Examples include use of bidirectional promoters or use of an internal ribosomal entry site (IRES) of positive-stranded RNA-viruses (e.g. derived from EMCV) to produce a single transcript that is translated into two proteins (e.g. (Amendola, Venneri, Biffi, Vigna, & Naldini, 2005; Na & Fan, 2010)). Other examples include utilizing the host cell splicing machinery or use of “cleavage” peptides derived from positive-stranded RNA viruses such as the foot-and-mouth-disease 2A sequence or equivalents from other viruses to produce a polyprotein that is cleaved into two proteins. According to published reports, all of these strategies can be equally useful and successful.
When two antigens are encoded in one adenoviral vector, several features of a monovalent vector should be maintained in order to make the multivalent vector both produceable and useful for vaccine purposes. Important features include genetic stability during upscaling, productivity of the vector at large scale, high level expression of both antigens, and immunogenicity of both antigens. However, for most of the published strategies the genetic stability and other features of the vectors have not been systematically analyzed.
Described herein are experimental results showing that approaches described in the prior art for expressing two antigens with a single recombinant adenovirus, lead to: a) reduced genetic stability in the upscaling process of the recombinant adenovirus (as can be mimicked by serial passaging in the helper cell line); b) reduced productivity of the recombinant adenovirus (decreasing the possibility to upscale the vectors to large purified batches); c) reduced expression of the antigens; and/or d) reduced immunogenicity of one or more of the antigens (in mouse model). These are clear disadvantages that do not support large scale use of recombinant adenovirus for expressing two antigens as described in the prior art.
Therefore, a need remains to provide a recombinant adenovirus that is genetically stable and that expresses two antigens in a manner in which the immunogenicity of both antigens is maintained.